Microfluidic flow assay for measuring hemostatic phenotypes

ABSTRACT

A microfluidic-based flow assay and methods of manufacturing the same are provided. Specifically, the microfluidic flow assay includes a micropatterned surface that induces clot formation and an array of microfluidic channels though which blood flows. The micropatterned surface contains two clotting stimuli, one for inducing platelet adhesion and another for inducing the coagulation cascade.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/313,257, filed Mar. 12, 2010, the entiredisclosure of which is hereby incorporated herein by reference.

FIELD OF INVENTION

The invention relates to a microfluidic-based flow assay for use inanalyzing bleeding and thrombotic disorders, dosing anticoagulant andantiplatelet drugs, tracking the effects of pharmacologicalinterventions on thrombosis, and methods of making the same.

BACKGROUND OF INVENTION

Maintaining the balance between bleeding and thrombosis remains one ofthe greatest challenges facing the biomedical community. Excessivebleeding is an important medical issue. For example, post partumbleeding represents a leading cause of maternal mortality and causesserious morbidity in developing countries. Individuals with geneticbleeding disorders, such as hemophilia, have a decreased ability to clotblood because of deficiencies in certain coagulation factors.

On the other end of the spectrum, excessive clotting, or thrombosis, isa major complication of surgery and is integrally involved inatherosclerosis, obesity, infection, diabetes, cancer, and autoimmunedisorders. Over the last decade, significant advances have been made inunderstanding the molecular basis of bleeding and thrombotic disorders;however, a large portion of the observed variability remains unknown.

Parallel with these discoveries, there has been a rapid development ofnew drugs like recombinant proteins for replacement and interventionaltherapies. Interestingly, what remains strikingly deficient in clinicalhematology are techniques to diagnose a very broad range of disorders ofboth deficient and excessive clotting as well as to monitor the effectsof therapeutic interventions.

Diagnosing the severity of bleeding disorder is impossible with currentbleeding assays, particularly because most current bleeding assays testfor either platelet function or coagulation, but not both. Thus, mostexisting solutions do not properly create an environment which properlysimulates a natural human wound or point of bleeding. In addition, mostof these conventional assays occur under static, or no flow, conditions.Since blood is a moving fluid, however, there are several advantages tostudying it under flow in bleeding diagnostics.

SUMMARY OF INVENTION

It is, therefore, one aspect of the present invention to provide adevice which contains two clotting stimuli, one for inducing plateletadhesion and another for inducing the coagulation cascade.

It is another aspect of the present invention to provide a device whichallows blood to flow over a micro-patterned surface which induces clotformation. In some embodiments, a microfluidic channel is provided withone or more clot inducing areas. Each of the one or more clot inducingareas may include a micro-patterned surface that induces clot formationvia two different stimuli (e.g., inducing platelet adhesion and inducingcoagulation).

It is another aspect of the present invention to combine the physics ofblood flow and the biology of the clotting system into a single device.

In accordance with at least some embodiments of the present invention, amicrofluidic flow assay is provided which accounts for the three mainfactors which contribute to the formation of a blood clot: platelets,coagulation, and blood flow.

Platelets are the first responders to a vascular injury. A vascularinjury can be due to trauma or the rupture of an atherosclerotic plaque.Platelets adhere to proteins, especially collagens, found underneath thecells that line blood vessels and von Willebrand factor, which issecreted by endothelial cells and platelets. Following plateletadhesion, a series of enzymatic reactions occur that are collectivelyknown as the coagulation cascade. The main catalyst for the coagulationcascade is a transmembrane protein called tissue factor.

Embodiments of the present invention provide a microfluidic devicehaving a clot inducing area in a microfluidic channel though which bloodis allowed to flow, where the clot inducing area includes a mixture ofcollagen, von Willebrand factor, and tissue factor. In some embodiments,the area(s) of tissue factor which are exposed to blood flowing therebyare interspersed in the collagen in a predetermined pattern.

Because there is significant variability in clotting factors and bloodcell counts in the healthy population, it is useful to provide amicrofluidic device in which the microfluidic channel(s) and clotinducing areas, which are also referred to as prothrombotic surfaces,are homogeneous and repeatable. Otherwise, it may become difficult todetermine whether differences in platelet and fibrin accumulation arevariations in blood constituent or variability in the prothromboticsurface. It is, therefore, another aspect of the present invention tostandardize the methods for patterning molecules that stimulate thesetwo mechanisms and evaluate the microfluidic flow assay in a clinicalsetting. More specifically, embodiments of the present invention providea homogeneous, repeatable collagen patterning method for measuringplatelet adhesion. Embodiments of the present invention also provide arepeatable method for co-patterning collage and tissue factor formeasuring coagulation defects.

It is another aspect of the present invention to provide a flow assaywhich allows the in vitro study of platelet response to defined surfacesat controlled wall shear stresses (e.g., via use of a microfluidicchannel).

In accordance with at least some embodiments of the present invention, amicrofluidic device is provided which generally comprises:

at least one microfluidic channel; and

at least one prothrombotic surface provided in the at least onemicrofluidic channel, wherein the at least one prothrombotic surface iscapable of inducing both platelet adhesion and coagulation cascade.

In accordance with at least some embodiments of the present invention, amethod of manufacturing a microfluidic device is provided whichgenerally comprises:

providing a substrate;

creating at least one prothrombotic surface on the substrate, whereinthe at least one prothrombotic surface is capable of inducing bothplatelet adhesion and coagulation cascade; and

establishing at least one microfluidic channel which intersects at leasta portion of the at least one prothrombotic surface.

In accordance with at least some embodiments of the present invention, amicrofluidic device made by the above-described method is also provided.

In accordance with at least some embodiments of the present invention, amicrofluidic channel through which blood is capable of flowing isprovided that generally comprises:

at least one prothrombotic surface provided as a part of at least aportion of one surface in the channel, wherein the at least oneprothrombotic surface is capable of inducing both platelet adhesion andcoagulation cascade in the blood.

In accordance with at least some embodiments of the present invention, akit for measuring clot formation is provided which generally comprises:

a vacuum or hemetically sealed microfluidic device, the microfluidicdevice comprising at least one microfluidic channel and at least oneprothrombotic surface provided in the at least one microfluidic channel,wherein the at least one prothrombotic surface is capable of inducingboth platelet adhesion and the coagulation cascade.

In accordance with at least some embodiments of the present invention, amethod of measuring clot formation or clotting characteristics isprovided which generally comprises:

causing blood to flow through a microfluidic channel under laminar flowconditions, wherein the blood flows through the microfluidic channel andacross at least one prothrombotic surface provided in the microfluidicchannel, wherein the at least one prothrombotic surface is capable ofinducing both platelet adhesion and coagulation cascade; and

analyzing, around the at least one prothrombotic surface, a number ofblood cells which have substantially stopped flowing.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a top view of an exemplary microfluidic device inaccordance with at least some embodiments of the present invention;

FIG. 2 a depicts an exploded top view of a portion of an exemplarymicrofluidic channel in accordance with at least some embodiments of thepresent invention;

FIG. 2 b depicts an exploded partial cross-sectional view across line2-2 of an exemplary microfluidic channel in accordance with at leastsome embodiments of the present invention;

FIG. 3 depicts an exemplary method of manufacturing a microfluidicdevice in accordance with at least some embodiments of the presentinvention;

FIG. 4 depicts a partial cross-sectional view of a microfluidic channelat a first step of manufacturing in accordance with at least someembodiments of the present invention;

FIG. 5 depicts a partial cross-sectional view of a microfluidic channelat a second step of manufacturing in accordance with at least someembodiments of the present invention;

FIG. 6 depicts a partial cross-sectional view of a microfluidic channelat a third step of manufacturing in accordance with at least someembodiments of the present invention;

FIG. 7 depicts a partial cross-sectional view of a microfluidic channelat a fourth step of manufacturing in accordance with at least someembodiments of the present invention;

FIG. 8 depicts a partial cross-sectional view of a microfluidic channelat a fifth step of manufacturing in accordance with at least someembodiments of the present invention; and

FIG. 9 depicts a partial cross-sectional view of a microfluidic channelat a sixth step of manufacturing in accordance with at least someembodiments of the present invention.

DETAILED DESCRIPTION

Embodiments of the present invention will now be described in connectionwith methods, devices, and systems used for testing blood clotting ordetermining whether an individual is prone to blood-clotting issues.However, those skilled in the art will appreciate that embodiments ofthe present invention are not limited to the field of blood flow and canbe utilized in other fields without departing from the scope of thepresent invention.

Referring initially to FIG. 1, an exemplary microfluidic device 100 willbe described in accordance with at least some embodiments of the presentinvention. More specifically, the microfluidic device 100 may include aplurality of fluid-receiving passages 104 a, 104 b, 104 c, which arecapable of receiving fluid at a receiving end 108 and allowing saidfluid to flow through a microfluidic channel 116 to a terminal end 112.

In accordance with at least some embodiments of the present invention,the fluid flowing through the microfluidic channel 116 may be blood,such as human blood. Additional details of microfluidic channels whichfacilitate laminar flow conditions are described, for example, in U.S.Pat. No. 7,318,902 to Oakey et al., the entire contents of which arehereby incorporated herein by reference.

Although three receiving ends 108 are depicted, one skilled in the artwill appreciate that one or more of the microfluidic channels 116 maysplit into multiple channels, thereby resulting in a number of terminalends 112 which exceeds the number of receiving ends 108. As can beappreciated by one skilled in the art, however, the number of receivingends 108 may equal the number of terminal ends 112. The configurationand design of the microfluidic channels 116 can vary without departingfrom the scope of the present invention.

In addition to comprising microfluidic channels 116, the microfluidicdevice 100 may also comprise one or more prothrombotic structures 120 a,120 b which intersect one or more of the microfluidic channels 116. Inparticular, a prothrombotic structure 120 a, 120 b include a first end124, a second end 128, and a prothrombotic surface 132 therebetween. Theprothrombotic surface 132 may intersect the microfluidic channel 116 atan area of intersection generally referred to as a clot forming area136. This clot forming area 136 is an area within the microfluidicchannel 116 in which both platelet adhesion and coagulation cascade isinduced in the blood flowing through the microfluidic channel 116.

The specific properties of the prothrombotic surface 132 which induceboth platelet adhesion and coagulation cascade will now be described inconnection with FIGS. 2 a and 2 b. In particular, the clot forming area136 is depicted in further detail in FIGS. 2 a and 3 b. In accordancewith at least some embodiments of the present invention, theprothrombotic surface 132 includes collagen 212 or a similar materialknown to induce platelet adhesion. The prothrombotic surface 132 mayalso include structures of tissue factor 216, which are designed toinduce the coagulation cascade. By providing the prothrombotic surface132 with both collagen 212 and tissue factor 216, the prothromboticsurface 132 is capable of inducing both platelet adhesion andcoagulation cascade in blood flowing through the microfluidic channel116 when the blood traverses the clot forming area 136.

Surrounding the clot forming area 136 in the microfluidic channel 116are a first 204 and second 208 passive surface, which may or may notinclude endothelial cells, and which is generally neutral with respectto inducing blood clotting. Accordingly, the amount of blood clottinginduced within the microfluidic channel 116 can be tightly controlled byprecisely controlling the size of the prothrombotic surface 132 and theamount of tissue factor 216 provided therein.

In accordance with at least some embodiments of the present invention,the first 204 and second 208 surface may be considered a neutral orpassivated surface. In some embodiments, a lipid is used for the first204 and second 208 surface. In particular, a bovine serum albumin (BSA)may be utilized as a passivity protein. This particular protein is knownnot to induce any type of blood clotting, such as platelet adhesion orthe coagulation cascade.

The width of the prothrombotic surface 132 may vary depending upondesired clotting or the size of the microfluidic channel 116 (e.g.,cross-sectional area of the microfluidic channel 116). In someembodiments, the width of the prothrombotic surface 132 may be about 100microns. This may be a particularly useful size of prothrombotic surface132 if the microfluidic channel 116 comprises a cross sectional area ofabout 50 microns×250 microns. This particular geometry is useful becauseit provides an area of constant shear stress across the middle of thechannel 116. As can be appreciated by one skilled in the art, however,the actual width of the prothrombotic surface 132 can have a greater orlesser size without departing from the scope of the present invention.

The structures of tissue factor 216 may comprise any type of shape. Forexample, although the structures of tissue factor 216 are depicted ashaving a generally circular cross-section, the structures of tissuefactor 216 may comprise a square cross-section, oval cross-section,rectangular cross-section, or unshaped cross-section. In someembodiments, a circular cross-section tissue factor 216 may comprise adiameter of between 10-100 microns. Smaller or larger structures oftissue factor 216 may be used. It should be noted that a 10 microndiameter island of tissue factor 216 substantially represents a singlecell in a human. Thus, utilization of a structure of tissue factor 216having a diameter of about 10 microns may be preferable for modeling atypical human bleeding environment. Additionally, the number ofstructures of tissue factor 216 provided in the clot forming area 136can be any number larger than one and the distribution of the structuresof tissue factor 216 within the collagen can either be symmetrical,asymmetrical, or random. As one exemplary distribution, lanes of tissuefactor 216 may be provided in the clot forming area 136 that traversesubstantially the length of the clot forming area 136 but do nottraverse the width of the clot forming area 136. The lanes of tissuefactor 216 may be separated by non-tissue factor lanes.

Moreover, the ratio of collagen 212 surface area to tissue factor 216surface area in a given prothrombotic surface 132 is less than 1:1. Inmore preferred embodiments, there is at least a 2:1 ratio of collage 212surface area to tissue factor 216 surface area, meaning that for everysquare nm of tissue factor 216 exposed there is at least two square nmof collagen 212 exposed. This ratio can also vary according toconditions and the size of the microfluidic channel 216 withoutdeparting from the scope of the present invention.

In accordance with at least some embodiments of the present invention,the collagen 212 is constructed of a fibrillar type 1 collagen. In someembodiments, an equine or rat tail-based collagen can be used. Incertain embodiments where a tighter control on variables within themicrofluidic device 100 is required, an acid soluble collagen having amore homogeneous surface than a non-acid soluble collagen can beutilized. For example, rat tail digested in a pH 3 environment can beutilized as a purer form of collagen than a non-treated collagen.

In accordance with at least some embodiments of the present invention,the structures of tissue factor 216 can be constructed of a lipid orlipid membrane that is used as an expressed surface of “activated”cells. This creates a significant amount of a molecule known as thrombinwhen blood plasma interacts with the lipid. Thrombin is a known serineprotease that creates a biopolymer of fibrin by cleaving fibrinopeptidefrom the plasma protein fibrinogen. Fibrin forms a highly entangledhydrogel that provides the scaffold onto which a blood clot grows.Generally speaking, high concentrations of thrombin are created duringvia the extrinsic or tissue factor pathway of the coagulation cascade;this is why tissue factor is known as a coagulation cascade inducingagent.

As can be seen in FIG. 2 b, the first 204 and second 208 areas ofneutral material as well as the prothrombotic surface 132 (comprisingthe layer of collagen 212 and structures of tissue factor 216) may beprovided on a substrate 218. Additionally, the microfluidic channel 116may be enclosed with a lid or top layer 220. In some embodiments thesubstrate 218 and/or lid or top layer 220 is constructed ofpolydimethylsiloxane (PDMS) or a similar type of silicone. As analternative, or in addition, the substrate 218 and/or lid or top layer220 is constructed of glass, plastic, gold, combinations thereof, or anyother type of known substrate material used in surface chemistry.

Referring now to FIGS. 3-9, an exemplary method of constructing amicrofluidic device 100 will be described in accordance with at leastsome embodiments of the present invention. The method begins byproviding a substrate 218 (FIG. 4; step 304). Thereafter, the substrate218 is functionalized (step 308). In this step, the substrate 218 may betreated with octadecyltrichlorosilane (OTS), thereby creating amonolayer of OTS on the upper surface of the substrate 218. Methods ofrendering substrates, such as glass substrates, hydrophobic are wellknown in the art. Methods of functionalizing the substrate 218 include,without limitation, rendering the substrate hydrophobic, hydrophilic,reactive (via amine or carboxylic acid groups), or some other chemistry.In particular, different surface chemistries may allow differentmolecules to be patterned in a specific configuration. In oneembodiment, silane chemistries may be used on glass substrates.

After the substrate 218 has been functionalized, the method continues byforming the prothrombotic surface 132, which will ultimately include theclot forming area 136 (FIG. 5; step 312). In particular, the BSA may beprovided as the first 204 and second 208 surface. The void between thefirst 204 and second 208 surfaces generally corresponds to the channelin which the prothrombotic structure 120 a or 120 b will be created.This void may be created by providing a masking layer on the substrate218 prior to applying the BSA to the substrate 218. After the BSA hasbeen provided, the masking layer may be removed from the substrate 218,thereby exposing the void between the first 204 and second 208 areas.

After the BSA has been laid down on the substrate 218, the methodcontinues by positioning a pillar-creating structure 604 on thesubstrate 218, particularly in the clot forming area 136 (FIG. 6; step316). This pillar-creating structure 604 may comprise a PDMS structurethat is formed to have pillars or structures of a size substantially thesame as a desired size of the structures of tissue factor 216. In someembodiments, the pillar-creating structure 604 comprises a singlestructure having a plurality of posts extending therefrom which touchthe surface of the substrate 218 in the clot forming area 136. In otherembodiments, the pillar-creating structure 604 comprises a plurality ofdistinct posts placed on the surface of the substrate 218 in the clotforming area 136. The posts of the pillar-creating structure 604 aregenerally used as masks to prevent collagen from adhering to thesubstrate 218 during subsequent manufacturing steps.

Once the pillar-creating structure 604 is in place, the method continuesby adding a collagen material 212 to the substrate 218 in the clotforming area 136 (FIG. 7; step 320). The collagen material 212 interactswith the exposed surface of the substrate 218 and eventually adheresthereto. Also, the collagen material 212 does not adhere to thesubstrate 218 in areas where the pillar-creating structure 604 ispresent. In some embodiments, the collagen material 212 may be added tothe substrate 218 by submerging the substrate 218 in a collagen bath fora sufficient time to ensure that an even layer of collagen material 212has been created in the clot forming area 136.

Thereafter, the method continues by removing the pillar-creatingstructure 604 from the substrate 218 to reveal collagen voids 804 (FIG.8; step 324). The lipid 216 is then added to the substrate 218 to fillthe collagen voids 804, thereby creating the structures of tissue factor216 (FIG. 9; step 328). Again, the lipid 216 can be added to thesubstrate 218 by submerging the substrate 218 into a lipid bath untilthe collagen voids 804 have been sufficiently filled and a substantiallysmooth lower surface of the microfluidic channel 116 has been created(e.g., substantially smooth surface is created between the first area204 of BSA, the collagen material 212, the structures of tissue factor216, and the second area 208 of BSA).

At this point, the creation of the prothrombotic structure 120 a or 120b is complete. As can be appreciated by one skilled in the art, althoughthe method of creating only one prothrombotic structure 120 a or 120 bwas depicted and described, multiple prothrombotic structures 120 a and120 b (or more) can be created at substantially the same time on asingle substrate 218 by following the steps described above.Accordingly, multiple prothrombotic structures 120 a and 120 b can becreated at substantially the same time, thereby reducing the amount oftime required to construct a microfluidic device 100.

As an alternative to creating pillar structures 604, embodiments of thepresent invention are capable of utilizing laminar flow patterning inwhich both protein and lipid solutions are introduced simultaneouslyunder flow to the substrate 218 to form lanes (i.e., alternating lanesof protein-lipid-protein-lipid-protein-etc. across the width of the clotforming area 136) Accordingly, rather than creating pillar structures inthe clot forming area 136, lane structures that have a long axissubstantially parallel to the fluid flow direction are created. In theevent that laminar flow patterning is employed to create lanestructures, the laminar flow patterning step may replace or augment oneor more of steps 316, 320, 324, and 328.

Once the prothrombotic structure(s) 120 a and/or 120 b are in place, themethod continues by positioning a microfluidic channel structure or lid220 across the clot forming area(s) 136 (FIG. 9; step 332). As discussedin connection with FIG. 1, the microfluidic channels 116 may be createdwithin a PDMS material and may be positioned to intersect theprothrombotic structure(s) 120 a and/or 120 b at only selectedlocations, thereby controlling the amount of clot forming that isinduced when blood is flowed through the microfluidic channels 116.

At this point, a microfluidic device 100 has been created. In accordancewith at least some embodiments of the present invention, themicrofluidic device 100 may be hygienically sealed in a sterileenvironment (e.g., hermetic plastic package) (step 336) such that themicrofluidic device 100 can be distributed as a clot testing kit tomedical personnel and other interested parties (step 340). One possiblecomplication with a kit that may need to be addressed is the fact thatlipids must be stored in an aqueous environment, in other words, theycan't be dried out. Accordingly, prior to hermetically sealing themicrofluidic device 100 in a sterile environment, an aqueous solutionmay be injected into the hermetic packaging prior to final sealing. Thiswill enhance the shelf life of the kit as well as enhance its ability tobe distributed great distances away from its source of manufacture.

In other embodiments, a vacuum source can be utilized to vacuum seal thesubstrate 218 to the lid 220. Such a device 100 can be used in varioustesting facilities. Moreover, the vacuum assisted sealing of the lid 220and substrate 218 is a reversible bonding technique which may allowtesting personnel to reposition the lid 220 relative to the substrate218 without damaging either component.

In accordance with at least some embodiments of the present invention,once the microfluidic device 100 has been prepared, one or more bloodsamples can be passed through the microfluidic channels 116 of thedevice 100 to analyze bleeding and thrombotic disorders, dosinganticoagulant and antiplatelet drugs, tracking the effects ofpharmacological interventions on thrombosis, and the like. Inparticular, platelet flow can be analyzed and platelet adhesion can bequantified in any number ways. As one example, platelet labeling anddetection schemes can be employed whereby blood platelets arefluorescently labeled with a small molecule or platelet specificantibody that is fluorescently visible. As the blood with the labeledplatelets flows through the microfluidic channels 116 of the device 100,and more specifically across the clot forming areas 136 in the channels116, the number (specific or relative) of platelets that have adhered toand/or around the clot forming area 136 may be observed. Suchobservations can be made in real-time with fluorescent imaging devices,cameras, recording devices, and the like, or after an experiment hasbeen performed. Real-time and/or post-testing analysis can help yieldquantifiable results as to the number of platelets that have adhered tothe clot forming area 136, which can then be correlated to standard testresults or other variables to determine whether the patient is prone toexcessive bleeding or the like.

The foregoing description of the present invention has been presentedfor purposes of illustration and description. Furthermore, thedescription is not intended to limit the invention to the form disclosedherein. Consequently, variations and modifications commensurate with theabove teachings, and the skill or knowledge of the relevant art, arewithin the scope of the present invention. The embodiments describedhereinabove are further intended to explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

1. A microfluidic device, comprising: at least one microfluidic channel;and at least one prothrombotic surface provided in the at least onemicrofluidic channel, wherein the at least one prothrombotic surface iscapable of inducing both platelet adhesion and coagulation cascade. 2.The device of claim 1, wherein the at least one prothrombotic surfaceincludes collagen which induces the platelet adhesion.
 3. The device ofclaim 2, wherein the at least one prothrombotic surface includes tissuefactor which induces the coagulation cascade.
 4. The device of claim 3,wherein the tissue factor is interspersed in a predetermined geometrythroughout the collagen.
 5. The device of claim 4, wherein a surfacearea of the at least one prothrombotic surface is at least ten timessmaller than a surface area of the at least one microfluidic channel. 6.The device of claim 4, wherein a surface area of the at least oneprothrombotic surface is at least one hundred times smaller than asurface area of the at least one microfluidic channel.
 7. The device ofclaim 4, wherein a plurality of tissue factor islands are provided inthe collagen and wherein each of the tissue factor islands have asurface area of less than 100 microns.
 8. The device of claim 1, whereinthe at least one prothrombotic surface comprises a surface area of lessthan 10,000 square microns.
 9. A method of manufacturing a microfluidicdevice, comprising: providing a substrate; creating at least oneprothrombotic surface on the substrate, wherein the at least oneprothrombotic surface is capable of inducing both platelet adhesion andcoagulation cascade; and establishing at least one microfluidic channelwhich intersects at least a portion of the at least one prothromboticsurface.
 10. The method of claim 9, wherein the at least oneprothrombotic surface includes collagen which induces the plateletadhesion.
 11. The method of claim 10, wherein the at least oneprothrombotic surface includes tissue factor which induces thecoagulation cascade.
 12. The method of claim 11, wherein the tissuefactor is interspersed in a predetermined throughout the collagen. 13.The method of claim 11, wherein creating the at least one prothromboticsurface comprises: adding collagen to the prothrombotic surface;providing a neutral material on the substrate and around the collagen;and interspersing a lipid in the collagen, wherein the lipid is providedin a predetermined pattern within the collagen and wherein the lipidcomprises the tissue factor.
 14. The method of claim 13, wherein thecollagen comprises an acid soluble collagen.
 15. A microfluidic devicemade by a method, the method comprising: providing a substrate; creatingat least one prothrombotic surface on the substrate, wherein the atleast one prothrombotic surface is capable of inducing both plateletadhesion and coagulation cascade; and establishing at least onemicrofluidic channel which intersects at least a portion of the at leastone prothrombotic surface.
 16. A microfluidic channel through whichblood is capable of flowing, the channel comprising: at least oneprothrombotic surface provided as a part of at least a portion of onesurface in the channel, wherein the at least one prothrombotic surfaceis capable of inducing both platelet adhesion and coagulation cascade inthe blood, wherein a surface area of the at least one prothromboticsurface is at least one hundred times smaller than a surface area of theat least one microfluidic channel, wherein a plurality of tissue factorislands are provided in the collagen and wherein each of the tissuefactor islands have a surface area of less than 100 microns, wherein theat least one prothrombotic surface includes tissue factor which inducesthe coagulation cascade, and wherein the at least one prothromboticsurface includes collagen which induces the platelet adhesion.
 17. A kitfor measuring clotting characteristics of blood, the kit comprising: ahermetically sealed microfluidic device, the microfluidic devicecomprising at least one microfluidic channel and at least oneprothrombotic surface provided in the at least one microfluidic channel,wherein the at least one prothrombotic surface is capable of inducingboth platelet adhesion and coagulation cascade.
 18. A method ofassessing clot characteristics of blood, comprising: causing blood toflow through a microfluidic channel under laminar flow conditions,wherein the blood flows through the microfluidic channel and across atleast one prothrombotic surface provided in the microfluidic channel,wherein the at least one prothrombotic surface is capable of inducingboth platelet adhesion and coagulation cascade; and analyzing, aroundthe at least one prothrombotic surface, a number of blood cells whichhave substantially stopped flowing.